I. Interleukin-5 and the Humoral Response to Microbial Infection
Antibodies are produced by the immune system of humans and animals in response to the presence of molecules that are considered "foreign." Such molecules fall into two classes: "antigens" or "haptens." An antigen is a molecule whose presence in an animal is capable of inducing the immune system to produce antibodies. In contrast, a hapten is capable of being bound by an antibody, but is not capable of eliciting antibody formation. Haptens are generally small molecules; when conjugated to a larger molecule, they can become antigens, and thus induce antibody formation. The nature and structure of antibodies, and the tenets of immunology are disclosed by Davis, B. D. et al. (In: Microbiology, 2nd Ed., Harper & Row, NY (1973)).
The capacity to induce antibody formation in response to microbial infection depends upon a series of interactions among T cells, B cells, and macrophages. Each B cell is genetically programmed to produce cells that express an immunoglobulin (or antibody) that is capable of specific interaction with a distinct antigenic determinant on the infectious microbe. Such antibodies play a central role in the humoral response to infection (Takatsu, K., Microbial Rev 35:593-606 (1991)).
The B cell response to an antigen is regulated by a helper T cell responding to, and specific for, the same antigen molecule (Takatsu, K., Microbial Rev 35:593-606 (1991)). The helper T cells recognize antigenic peptides in the context of class II major histocompatability complex (MHC) molecules that are arrayed on B cells, and secrete several soluble factors ("lymphokines") which can induce growth and maturation of B cells (Howard, M. et al., Ann. Rev. Immunol. 1:307-333 (1983); Kishimoto, T. et al., Ann. Rev. Immunol. 3:133-157 (1985); Melchers, F. et al., Ann Rev. Immunol. 4:13-36 (1986); Takatsu, K., Microbial Rev 35:593-606 (1991)).
One of these soluble factors (termed, "T-cell-replacing factor" ("TRF") was found to induce the terminal differentiation of activated B cells into antibody producing cells (Takatsu, K. et al., J. Immunol. 124:2414-2422 (1980)). This factor was subsequently found to have an activity (termed "BCGF II") that could promote DNA synthesis in certain B cell leukemic cells (Harada, N. et al., J. Immunol. 134:3944-3951 (1985)). Further research revealed the additional presence of an eosinophil differentiation factor capable of inducing eosinophil colony formation (Sanderson, C. J. et al., J. Exper. Med. 162:60-74 (1985)). Ultimately, these factors were purified (Sanderson, C. J. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:437-440 (1986)) and sequenced (Campbell, H. D. et al., Eur. J. Biochem, 174:345-352 (1988); Kinashi, T. et al., Nature 324:70-73 (1986)), and found to be derived from a single protein, termed "interleukin-5" ("IL-5") (McKenzie, A. N. N. et al., In: Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel, Karger, vol. 51, pp 135-152 (1992), herein incorporated by reference).
The cDNA encoding murine IL-5-TRF/BCGFII was cloned by using SP6 expression vector system (Kinashi, T., et al., Nature 324:70-73 (1986)). The amino acid sequence deduced from the nucleotide sequence of the entire IL-5 cDNA revealed that murine IL-5 consists of 133 amino acids including a hydrophobic signal sequence of 20 amino acids (Kinashi, T., et al., Nature 324:70-73 (1986)) with a molecular mass of 12.3 kDa. Thus, the mature human IL-5 protein has 113 amino acids, and is the same length as the mature murine IL-5. Comparison of the cDNA sequence of murine IL-5 with that of human shows a sequence homology of 77% at the DNA level and 70% at the protein level.
Interleukin-5 is a homodimeric glycoprotein. Investigations using reduced and alkylated IL-5 suggest that dimerization is essential for biological activity (Tsuroka, N., et al., Cell. Immunol. 125:354-362 (1990)). Comparison of the two polypeptide sequences shows 81 identical amino acids, 25 conservative amino acid changes, with the remaining 7 (and the two N-terminal amino acids of the human protein sequence) as non-conservative changes. This high degree of similarity is reflected in the ability of the two proteins to cross-react with cells of other mammals (Sanderson, C. J., et al., In: Colony Stimulating Factors: Molecular and Cellular Biology, Marcel Dekker, NY, pp 231-256 (1990)). Despite their capacity to cross react, murine and human IL-5 display significant species specificity, with the murine material being approximately 100-fold more active against murine cells than the human molecule. Conversely, human IL-5 is approximately 20-fold more active in human bone marrow assays than murine IL-5)McKenzie, A. N. J. et al., In: Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel, Karger, vol. 51, pp 135-152 (1992)).
Native IL-5 or recombinant IL-5 expressed in mammalian cells in heterogeneously glycosylated, with the mouse sequence containing three potential N-glycosylation sites, while the human sequence lacks on of these. Carbohydrate has been shown to be unnecessary for biological activity, although it is possible that it may perform some role in governing the half-life of the polypeptide in the circulation (McKenzie, A. N. J. et al., In: Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel, Karger, vol. 51, pp 135-152 (1992)).
Interleukin-5 promotes the growth of B-lineage cells. IL-5 acts on naturally activated B cells, on LPS-stimulated B cells, and on resting B cells to induce maturation and to propagate proliferation (Karasuyama, H. et al., J. Exper. Med. 167:1377-1390 (1988)). IL-5 induces the increase in levels of secreted forms of .mu.-mRNA in BCL.sub.1 or resting as well as activated B cells (Webb, C. P. et al. J. Immunol. 143:3934-3939 (1989)). Murine IL-5 was found to cause an increase in the frequency of B cells both proliferating and lg secreting (Alderson, M. R. et al., J. Immunol. 139:2656-2660 (1987)). IL-5 can induce antigen-specific and polyclonal IgA production in antigen-primed B cells and in LPS-stimulating B cells, respectively.
IL-5 also appears to promote immunoglobulin formation. Transgenic mice carrying the IL-5 gene exhibited elevated levels of IL-5 in the serum (2-10 ng/ml), and an increase in the levels of serum IgM and IgA. A marked increase in the number of peripheral blood white cells (PBL), of spleen cells and of peritoneal cells was also observed. Particularly, the increase in the numbers of eosinophils in PBL reached 70-fold those of age-matched control mice (Tominaga, A. et al., J. Exper. Med. 144:1345-1352 (1990)). Antibodies to IL-5 mAbs have been isolated (Coffman, R. L. et al., Science 245:308-311 (1989)), and have been found to inhibit the antigent-specific primary IgM response induced by a cloned helper T cells in an MHC-restricted manner and also to inhibit polyclonal Ig-secretion (Rasmussen, R. et al., J. Immunol. 140:705-712 (1988)).
Importantly, IL-5 also plays a role in the production and maturation of eosinophils. Eosinophils are immune system cells that accumulate in response to allergic inflammatory reactions. They ingest antibody-antigen complexes and thereby become degranulated. Since the granules contain substances capable of blocking the action of histamine, serotonin and bradykinin, all of which are involved in inflammation, it has been proposed that eosinophils protect the tissues of the host not only by phagocytizing and degrading cytotoxic antibody-antigen complexes but also by damping the effects of chemical mediators of the inflammatory response (Davis, B. D. et al., In: Microbiology, 2nd Edit., Harper & Row, NY (1973)). IL-5 thus plays an important part in diseases associated with increased iosiniphils, such as asthma and similar inflammatory conditions (Sanderson, C. J., In: Advances in Pharmacology, vol. 23, Academic Press, NY, pp. 163-177 (1992); Takatsu, K., Curr. Opin. Immunol. 4:299-306 (1992), both herein incorporated by reference).
Murine IL-5 has also been shown to induce the production of eosinophils in liquid bone marrow cultures (McKenzie, A. N. J. et al., In: Interleukins: Molecular Biology and Immunology, Chem. Immunol., Basel, Karger, vol. 51, pp 135-152 (1992)). maintain the viability of mature eosinophils, to induce the production of superoxide anion in mature eosinophils and to possess chemotactic activity for eosinophils (Harada, N. et al., J. Immunol. 134:3944-3951 (1985); Lopez, A. F. et al., J. Exp. Med. 167:219-224 (1987); Owen, W. F. et al., J. Exper. Med. 170:343-349 (1989); Yamaguchi, N. et al., J. Exper. Med. 167:43-56 (1988); Yamaguchi, N. et al., J. Exper. Med. 167:1737-1742 (1988)). The syngergistic effect of IL-5 and colony-stimulating factors on the expansion of eosinophils is supposed to contribute to the urgent mobilization of eosinophils at the time of helminthic infections and allergic responses.
Thus, IL-5 appears to play a significant role in inducing inflammatory processes. Molecules that potentiate this activity are therefore highly desired anti-inflammatory agents.
II. Antibodies and Immunoassays
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target molecule. Immunoassays are used extensively in modern diagnostics (Fackrell, J. Clin. Immunoassay 8:213-219 (1985)). A large number of different immunoassay formats have been described (Yolken, R. H., Rev. Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays, John Wiley & Sons, NY (1985); Ngo, T. T. et al., In: Enzyme Mediated Immunoassay, Plenum Press, NY (1985)).
The simplest immunoassay involves merely incubating an antibody that is capable of binding to a predetermined target molecule with a sample suspected to contain the target molecule. The presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule. In order to facilitate the separation of target-bound antibody from the unbound antibody initially present, a solid phase is typically employed. Thus, for example the sample can be passively bound to a solid support, and, after incubation with the antibody, the support can be washed to remove any unbound antibody.
In more sophisticated immunoassays, the concentration of the target molecule is determined by binding the antibody to a support, and then permitting the support to be in contact with a sample suspected to contain the target molecule. Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways. For example, the support can be incubated in the presence of a labelled, second antibody that is capable of binding to a second epitope of the target molecule. Immobilization of the labelled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample. In an alternative assay, the target is incubated with the sample and with a known amount of labelled target. The presence of any target molecules in the sample competes with the labelled target molecules for antibody binding sites. Thus, the amount of labelled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecule in the sample.
In general, immunoassay formats employ either radioactive labels ("RIAs") or enzyme labels ("ELISAs"). RIAs have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. Such assays suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipment. RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein.
ELISAs have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies--colorimetric, pH, gas evolution, etc.--can be used to quantitate the assay. In addition, the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use. ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D. M. et al., Eds.), John Wiley & Sons, NY (1988), incorporated by reference herein.
Antibodies that would be capable of specific binding to Interleukin-5 would be highly desired aids in detecting or quantifying IL-5 presence or levels. Although antibodies to the murine IL-5 have been identified that also bind human IL-5, it would be desirable to have antibodies of greater specificity and/or avidity to human IL-5. Such reagents would also be valuable in determining IL-5 derivatives and mimetics having increased or modified biological activity. The present invention provides such antibodies, as well as methods for their exploitation.